Metabolomic Study Of The Function Of Arabidopsis AZI1 In Systemic Acquired Resistance

Arabidopsis AZI1 (AZELAICACID INDUCED 1. AT4G12470) is a member of EARLI1 subfamily. The protein encoded by it is constituted by a N-terminal signal peptide, a hydrophilic proline-rich domain (PRD) in the middle and a C-terminal hydrophobic eight cysteine motif (8CM). Due to the existence of 8CM, AZI1 is also considered to be a member of the lipid transfer protein (LTP) family. AZI1 is named after its inducible expression by azelaic acid (AzA). Previous researches indicated that AZI1 was involved in defense response of Arabidopsis to pathogens such as Botrytis cinerea. In the present work, the wild-type Arabidopsis plants, the homozygous T-DNA knockout plants of AZI1 and the overexpressing plants of AZI1 were used as materials, and the function of AZI1 in systemic acquired resistance was identified by molecular biology and metabolomic methods.In experiments of systemic resistance analyses, three lower leaves in each Arabidopsis plant were first challenged with Pseudomonas syringae pv. maculicola ES4326 carrying the avirulence gene avrRPMl (Psm avrRPMl) (A) or 10 mmol/L MgSO4 (M), then the upper leaves were inoculated with virulent P. syringae pv. maculicola ES4326 (Psm ES4326) (V). Phenotypic observation showed that most of the upper leaves infected with Psm ES4326 remained green in AV treatment and most of the upper leaves infected with Psm ES4326 were necrotic in MV treatment in AZI1 overexpressing plants. In contrast, no significant difference could be found in AZI1 T-DNA knockout plants subjected to AV and MV treatments, and most of the upper leaves infected with Psm ES4326 were necrotic. Statistical analysis of the bacterium number in systemic leaves indicated that the virulent Psm ES4326 propagated much slower in AV-treated AZI1 overexpressing plants than in equivalent leaves of the wild-type plants and the homozygous AZI1 T-DNA knockout plants, further corroborating that AZI1 plays an important role in systemic defense of Arabidopsis against pathogens.RT-PCR analyses of the RNA samples extracted from leaves challenged with avirulent Psm avrRPM1 revealed that the expression levels of the pathogenesis related genes, including PR1, PR5 and GST1, were upregulated obviously in wild-type Arabidopsis plants, AZI1 T-DNA knockout plants and AZI1 overexpressing plants, indicating mutation of AZI1 did not influence the local resistance of Arabidopsis to pathogens. However, RT-PCR analyses of the RNA samples extracted from distal systemic leaves after localized foliar infections with avirulent Psm avrRPM1 showed that the transcription of PR1, PR5 and GST1 were significantly increased in AZI1 overexpressing plants, whereas the expression of these genes in AZI1 T-DNA knockout plants was reduced substantially or could not be detected, suggesting knockout of AZI1 can affect the systemic resistance of Arabidopsis to pathogens. RNA gel blot analysis revealed that the expression of PR1 in avirulent Psm avrRPM1-treated leaves of wild-type Arabidopsis plants was associated with the infection time. Along with the extension of infection time, the transcription level of PR1 was increased gradually.The distal systemic leaves were used as materials in metabolomic analysis by nuclear magnetic resonance (NMR) spectroscopy method and the contents of 34 kinds of metabolites in total were determined in wild-type Arabidopsis plants, AZ11 T-DNA knockout plants and AZI1 overexpressing plants after inoculation with avirulent Psm avrRPM1 or 10 mmol/L MgSO4. Principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) showed that obvious differences in the metabolic profiles could be detected between the avirulent pathogen-infected and the mock-treated plants. Multivariate statistical analysis ascertained that sucrose, glucose, fructose, sn-Glycero-3-phosphocholine, proline and glutamic acid were the main metabolites contributed to grouping, indicating glycometabolism and aminoacid metabolism are able to respond to biotic stresses and certain metabolic products of sugar probably are the signal molecules in systemic acquired resistance of Arabidopsis. Finally, the expression levels of the genes related with glycometabolism were analysed in distal systemic leaves after infection with avirulent pathogen or mock-treatment with 10 mmol/L MgSO4, and the results further confirmed that the "sweet immunity" had a function in systemic acquired resistance of Arabidopsis.